1. Field of the Invention
This invention relates to a novel protein-free maintenance medium for mammalian tumor cells. This invention further relates to a method of isolating tumor-secreted products using said maintenance medium.
2. Description of Related Art
Tumor-secreted products play critical roles in malignant growth. They subserve a multitude of functions including interference with the host immune response (North et al., J. Exp. Med. 143, 559-573 (1976); Veit et al., J. Immunol. 117, 655-660 (1976)), autostimulation of tumor cell growth (Sporn et al., New Engl. H. Med. 303, 878-880 (1980); Kaplan et al., Proc. Natl. Acad. Sci. USA 79, 485-489 (1982), modulation of invasion and metastasis (Goldfarb, R. N., in Tumor Invasion and Metastasis, eds. Liotta & Hart (Martinus Nijhoff, The Hague), pp. 375-390 (1982) and induction of neovascularization (Folkman, J., Adv. Cancer Res. 19, 331-358 (1974); Vallee et al., Experientia 41, 1-15 (1985)). The chemical and biological characterization of such molecules provide valuable insight into the mechanisms governing the complex biological processes of malignancy while also providing new means for cancer detection, diagnosis and therapeutic management. The identification as well as the structural and functional characterization of such molecules depends critically on the availability of systems from which such molecules can be isolated in high yield. While in vitro cultivation of established tumor lines remains the method of choice, it is laborious to purify tumor-secreted products to homogeneity from such cultures. This is due, in part, to the typical serum requirements of most cells, since its presence complicates the purification of such products.
The large-scale, in vitro cultivation of malignant cells can potentially produce tumor-derived biomolecules in yields sufficient for both biological and chemical characterization. However, for this purpose, exogenous serum and/or its growth factor components are virtually compulsory additives. Serum supplies growth factors, hormones, transport and attachment factors, or lipids whose nature, amounts or mechanisms of action remain largely unspecified. For example, Dulbecco's modified Eagle's medium (Whittaker M. A. Bioproducts, Walkersville, Md.) plus small amounts of glutamine, around 2 mM, has been studied in presence of serum and other proteins as a growth medium. Studies provide evidence that glutamine serves as a major energy source, not only for normal cells in vivo, but also for tumor cells cultivated in vitro. Energy provisions through glutamine utilization has been described for several tumor types which include HeLa, lymphoma and myeloma cells (Reitzer et al., J. Biol. Chem. 254, 2669-2676 (1979); Lavietes et al., Proc. Natl. Acad. Sci. USA 71, 3993-3997 (1974); Roberts et al., J. Cell Sci. 21, 609-615 (1976)). Moreover, increased glutamine utilization has been shown to correlate with malignant growth in vivo (Kovacevic et al., Cancer Res. 32, 326-333 (1972); Aaki et al., J. Biol. Chem. 257, 432-438 (1982)).
Techniques for cultivating both primary and established lines of mammalian tumor cells in serum-free but supplemented medium are well known. Such media can provide a suitable environment for cell proliferation but with a few exceptions (Healy et al., Proc. Soc. Exp. Biol. Med. 89, 71-77 (1955); Merchant et al., Proc. Soc. Exp. Biol. Med. 110, 194-198 (1962); Agy et al., In Vitro 17, 671-680 (1982); Kaighn et al., Proc. Natl. Acad. Sci. USA 78, 5673-5676 (1981) require supplementation with various hormones and proteins (Barnes et al., Anal. Biochem 102, 255-270 (1980)). Further, each new cell type usually requires an unique, defined medium for serum-free growth. The nutrient requirements of quiescent but actively metabolizing cells are typically much less than those needed for proliferation of cells (Ham, R. G., in Tissue Growth Factors, ed. Baserga, R. (Springer-Verlag, New York), pp. 13-74 (1981)).
Nevertheless, the addition of serum and other protein supplements has greatly complicated the procedure itself and, beyond that, the very objective, i.e., the identification and purification of tumor constituents which are usually present in vanishingly small amounts. Successful attempts as culturing tumor cells under serum-free conditions have been reported, but, while eliminating addition of serum, they usually have substituted such moieties obtained from it (Barnes et al., Anal. Biochem, 102, 255-270 (1980); Higuchi, K., Adv. Appl. Microbiol. 16, 111-136 (1973); Ham, R. G., in Tissue Growth Factors, ed. Baserga, R. (Springer-Verlag, New York, pp. 13-74 (1981)). Moreover, each cell line generally then requires a set of molecular species unique for its survival and devised to bring about cellular proliferation rather than maintenance of longterm viability and secretory capacity. As for normal cells, long-term survival in serum-free media has not been shown. By way of illustration, Hanks' Balanced Salt Solution (Whittaker M. A. Bioproducts, Walkersville, Md.) containing inorganic salts and glucose is used only as a transport medium to sustain normal cell viability and metabolism at reduced temperatures, around 4.degree. C., for extremely short periods of time, up to around 24 hours (Hanks et al., Proc.Soc.Exp. Biol. Med. 71, 196-200 (1949)). Other investigators have demonstrated survival of certain tumor cell lines or only 2-3 days with serum-free modified Eagle's medium (Iwata et al., Cancer Res. 45, 2689-2694 (1985); Moses et al., Cancer Res. 41, 2842-2848 (1981)). The ability to obtain long-term survival and retain secretory capacity has proven difficult, if not impossible, to achieve in protein-free media.
The addition of 2 mM, L-glutamine to serum-free Dulbecco's modified Eagle's medium containing 4.5 mg/mL of glucose, inter alia, has been described for use as a maintenance medium for HT-29 cell cultures in Fett et al., Biochemistry 24, 965-975 (1985), but this medium does not preserve both the long-term viability and firm attachment of cells in large-scale cultures.
Dvorak et al. U.S. Pat. No. 4,456,550 describes the use of Dulbecco's modified Eagle's medium having a glucose content of 4 g/l, inter alia, as a medium for Line 10 tumor cells, but the medium lacks L-glutamine. Tolbert et al. U.S. Patent No. 4,217,412 describes a culture medium of Dulbecco's modified Eagle's medium to which are added 4 mg/mL glucose and known essential amino acids, mineral salts, vitamins, and carbohydrates can be substituted for it in growing TE-671 cells; there is no mention of L-glutamine.
Alderman et al., Proc.Natl.Acad.Sci., USA 82, 5771-5775 (September, 1985) describes the medium of the present invention.
Unexpectedly, it has been found that 5 mM L-glutamine is sufficient to preserve both the long-term viability and firm attachment of cells in large-scale cultures that are free of exogenous protein, using the maintenance medium described herein, whereas 2 mM L-glutamine in the same medium is shown to be ineffective.